Disturbances of body water balance severely affect the quality of life and can lead to life-threatening conditions. Aquaporin (AQP) water channels play central roles in human water balance and are novel targets for pharmacological intervention.

To screen for blockers (aquaplugs) of AQPs, mammalian and yeast cell systems will be established. Inhibitors of aquaporin function will be identified in compound libraries and rationally designed. The activity, specificity and toxicity profile of these aquaplugs will be determined in optimised cell- and integrated systems. The atomic structures of AQPs and AQP-aquaplug complexes will be elucidated and used to design optimised aquaporin inhibitors. Studies will be performed to further determine the role of AQPs in organ water balance in health and disease to identify additional conditions where AQPs are targets for pharmaceutical application of aquaplugs. Identified aquaplugs with promising profiles will serve as lead structures for the development and exploitation of specific aquaporin inhibitors in novel skin care products and diuretics.

OBJECTIVES

The overall goal of this project is to establish the basis for the development of innovative skin care products and diuretics on the basis of inhibitors of aquaporin (AQP) function. To achieve these goals the research objectives are:

To establish test and screening systems for aquaporin blockers (aquaplugs) in yeast and mammalian cells.

To establish test systems in tissues and animals to test the function, specificity and potential toxicity of putative aquaplugs.

To determine and refine the structure of specific AQPs as a basis for further optimisation of aquaplug activity and specificity.

To determine the sites of interaction between AQPs and their inhibitors by structural and mutational analysis to further optimise the blockers.

To generate new knowledge on the physiological and pathophysiological role of aquaporins to identify further conditions where they could be targets for intervention.

The results of this project will be exploited through a world leader in skin care products and a new start-up company.

DESCRIPTION OF THE WORK

Alternative and complementary approaches at different levels are planned to achieve our goals:

Various cell based test and screening systems. Yeast and mammalian cell systems expressing different aquaporins (AQPs) will be established and used to:

Screen for compounds that block AQP function (aquaplugs).

Analyse their specificity and mode of action.

Various integrated test systems. In order to study the activity, selectivity and toxic effects of putative aquaplugs in integrated systems, ex vivo and animal model test systems will be established.

Various approaches to identify AQP blockers. Established yeast and mammalian cell systems will be used to screen for AQP blockers in compound libraries. Further, compounds will be rationally designed and selected on the basis of substrate analogues that can be transported through different AQPs when expressed in yeast.

Effects of aquaplugs in cells, tissue and animals. The established cell-based systems will be used to determine the activity, specificity and toxicity of identified putative AQP blockers. Promising candidates will be analysed in integrated systems.

Structure of aquaporins. The structure of AQPs at the atomic 3D level will be established in order to understand how AQP blockers function. This will be the basis for the rational design of optimised AQP blockers.

Structure-function relationships. Analysis of AQP-aquaplug complexes at the atomic level and functional analysis of AQP mutants with altered selectivity will validate the proposed AQP model. Additionally, spontaneous mutations in expressed AQPs will be identified in yeast test systems following the selection for transport of alternative substrates, which will provide information on structure-function relationships.

Other AQPs as new targets for aquaporin blockers. It will be tested whether AQP expression levels in different organs are increased under conditions of elevated water permeability. This information may identify new targets for pharmaceutical intervention with AQP blockers.

DELIVERABLES

The expected deliverables are:

Systems to identify and analyse AQP blockers, which support quality assessment of optimised aquaplugs in the process of product development.

Compounds that block the AQP function, which form the basis for novel skin care products and diuretics.

Knowledge of the structure, function, specificity and toxicity of AQPs, which is essential for the rational design of optimised aquaplugs and their application in health care.

Knowledge of the roles of AQPs and their validity as targets for aquaplugs.